EPITHELIAL H+ TRANSPORT--STRUCTURE OF H,K-ATPASE

这项工作的长期目标是了解H+的机制 哺乳动物胃粘膜壁细胞的分泌。这个 胃溃疡的病理生理与H，K-ATPase密切相关 活性，直接被奥美拉唑抑制，间接被H2抑制 对抗者。上皮细胞中H+转运的一个鲜为人知的方面是 三磷酸腺苷的标量能量转化为 矢量能量表现在H+梯度上。H，K-ATPase是一种独特的 这一机制的实验模型，很容易从 胃粘膜，在那里形成一百万倍的H+跨 胃上皮细胞。H，K-ATPase基因的克隆和序列分析 泵的主要结构。水病分析预测多发性 可以想象形成H+和K+孔的跨膜α-螺旋。 实验证实或反驳在模拟这些模型中很重要 机制是H，K-ATPase构象变化的知识 在运输过程中。在本研究中，H，K-ATPase的定位 壁细胞顶膜将用单抗进行探测 (MAb)。用免疫电子显微镜观察单抗的侧向性。特异症 H，K-ATPase的结构域。配体诱导(E_1-E_2)和刺激诱导 构象转换将使用单抗、胃泡、 和分离的壁细胞。这项研究将提供H+的直接证据 泵的拓扑学，将阐明构象转变对于 运输，将有助于解释H，K-ATPase电子 衍射数据，将通知定点突变，并将 有助于理解H+转运的分子机制。

The long term goal of this work is to understand the mechanism of H+ secretion by parietal cells of mammalian gastric mucosa. The pathophysiology of gastric ulcers is intimately related to H,K-ATPase activity, which is inhibited directly by omeprazole and indirectly by H2 antagonists. A poorly understood aspect of H+ transport in epithelia is the mechanisms by which the scalar energy of ATP is converted into vectorial energy manifested in a H+ gradient. The H,K-ATPase is a unique experimental model for this mechanism, being readily isolated from the gastric mucosa, where it forms a million fold gradient of H+ across the gastric epithelium. Cloning and sequencing of H,K-ATPase cDNA has revealed the primary structure of the pump. Hydropathy analysis predicts multiple transmembrane alpha-helices which conceivably form H+ and K+ pores. Experimental confirmation or refutation important in modelling these mechanisms is knowledge of H,K-ATPase conformational changes occurring during transport. In this study, the orientation of the H,K-ATPase in the parietal cell apical membrane will be probed with monoclonal antibodies (Mab). The sidedness of Mab and by immunoelectron microscopy. Epitopic domains of the H,K-ATPase. Ligand-induced (E1-E2) and stimulation-induced conformational transitions will be documented using Mab, gastric vesicles, and isolated parietal cells. The study will provide direct evidence for H+ pump topography, will clarify conformational transitions essential to transport, will aid in the interpretation of H,K-ATPase electron diffraction data, will inform site-directed mutagenesis, and will contribute to understanding of the molecular mechanism of H+ transport.